1. CP887679- 0 Use of Biomarkers in Patients with Acute Cardiovascular Disease Allan S. Jaffe, MD.* Consultant - Cardiology & Laboratory Medicine Professor of Medicine Mayo Clinic and Medical School Rochester, Minnesota *Dr. Jaffe is a consultant and receives research support from Roche, Dade Behring and Beckmann Coulter. He has been or presently is a consultant to Ortho Diagnostics, Sensera, Diadexus, Abbott, Hawaii Biotech and Tartagen.

2. CP1051812-1 Clinical Outcomes by Braunwald Class DaysDays DaysDays Primary Unstable Angina Class B Postinfarction Angina Class C Accelerated Angina Class I Subacute Angina at Rest Class II Acute Angina at Rest Class III PP PPSurvival Infarct free Infarct free without intervention Survival Infarct free Infarct free without intervention 0.970.97 0.910.91 0.530.53 0.960.96 0.880.88 0.720.72 0.940.94 0.860.86 0.350.35 0.970.97 0.890.89 0.550.55 0.890.89 0.800.80 0.370.37 JACC 25:1286, 1995

3. CP887679- 2 ECG Risk Factors in Patients with ACS Death or MI (%) Days JACC 36(3):970-1062, 2000 ST elevation and depression ST depression only ST elevation only T-wave inversion only No ST or T-wave change n=237 n=287 n=93 n=216 n=78 CP956024-12

4. CP887679- 3 Relationship of ECG Changes and FPA* FPA (nM) *JACC 18:898-903, 1991 100 nl <1.5

5. CP887679- 4 Long-Term Prognostic Value of Elevated cTnI in Unstable Angina % free from death and/or AMI Time from admission (days) Normal cTnI Log rank 6.63, P=0.01 Elevated cTnI Circulation. 1997;95:2053-2059

6. CP887679- 5 Antman, 19981.1 (0.4-3.5) Benamer, 199813.7 (3.9-48.3) Brisisc, 19988.0 (0.9-65.1) Cin, 199617.9 (5.2-61.2) Galvani, 19976.6 (1.3-32.3) Hamm, 199211.7 (3.2-42.6) Luscher, 19972.5 (1.3-4.8) Ohman, 19964.7 (1.7-12.7) Olatidoye, 1998156.1 (17.4-1,402) Ottani, 19976.6 (2.0-22.1) Rebuzzi, 199825.3 (5.2-123.2) Solymoss, 19972.4 (0.7-8.1) Stubbs, 19961.5 (0.5-4.2) Wu, 199531.5 (6.7-144.9) Antman, 19963.8 (1.8-8.03) Individual Studies of Patients with“Unstable Angina” Without ST Segment Elevation Peto OR & 95% CI (fixed) Pooled OR P<0.0001 0.20.11 51050 Favors lower risk Favors higher risk 4.8 (3.6-6.5)

7. CP887679- 6 Antman, 19963.82 (1.03-14.18) Benamer, 199813.68 (3.87-48.33) Brisisc, 19987.96 (0.97-65.25) Cin, 199617.91 (5.24-61.25) Galvani, 19976.55 (1.32-32.38) Hamm, 199211.71 (3.22-42.57) Luscher, 19975.93 (1.61-21.79) Olatidoye, 1998156.17 (17.39-1,402.09) Rebuzzi, 199825.27 (5.18-123.23) Solymoss, 19974.93 (0.72-33.19) Wu, 199531.52 (6.89-144.19) Individual Studies of Patients with “Unstable Angina” and Normal CKMB Values Peto OR & 95% CI (fixed) Pooled OR P<0.0001 0.20.11 51050 Favors lower risk Favors higher risk 11.83 (7.56-18.51)

8. CP887679- 7 cTnI in Patients with Unstable Angina Mortality at 42 days (% of patients) CP886232- 7 NEJM 335:1342-1349, 1996 Cardiac Troponin I (ng/mL) 8311741481345067 1.01.83.53.96.27.8Risk ratio ---0.5-6.71.2-10.61.3-11.71.7-22.32.6-23.095% CI 

9. CP887679- 8 Synergism of ECG and Elevated Troponins (PARAGON) CP1099500-8 JACC 41:376, 2003 6-mo death/ MI rate n=262 ST-depression P=0.20 n=125 n=259 n=174 n=57 n=82 P=0.13 P=0.15 Troponin– Troponin+ Troponin– Troponin+

10. CP887679- 9 FRISC-II – GUSTO IV (Death and MI) CP1142356-17 James et al: Am J Med 115:181, 2003 14 12 10 8 6 4 2 0 14 12 10 8 6 4 2 0 0.01 µg/L FRISC-II Death and MI at 30 days (%) 0.03 µg/L P=0.04 Below cutoffAbove cutoff 0.1 µg/L P=0.01 P=0.15 Cutoff 0.01 µg/L GUSTO-IV 0.03 µg/L P<0.001 0.1 µg/L P<0.001 Cutoff 11 80 17 74 30 61 66 521 111 476 201 386 2658093287464306441,9925,1232,5634,5523,4463,679

11. CP887679- 10 0.0 10.0 20.0 30.0 40.0 50.0 60.0 ABCDEGH Total CV, % ACS180 Centaur Immuno 1 Access Access2 Vidas Liaison RXL CS Opus Immulite Alpha Dx ECi AIA 21 IFCC Precision Study (Clinical Chem 2004)

12. CP887679- 11 CP1148152-7 cTnI Positives and cTnT Levels: An assay Comparison Int J Cardiol 93:113, 2004 Positive R-TnI (%) TnT levels (  g/L) R-TnI pos R-TnI neg R-TnI pos R-TnI neg 0.010.10.20.30.40.50.60.70.80.91.0 > 1,217 653 171 72 46 14 19 16 1 1 7 7 4 4 25 212 264 285 245 178 167 128 92 65 54 430 82

13. CP887679- 12 OR 4.55; 2.66-7.78 Rapid Troponin I Assay Outcomes in Relation to Troponin Values: The Issue of Assay Sensitivity CP1148152-9 Int J Cardiol 93:113, 2004 % % Neg Death MI Death or MI 56 9892130132205 Troponin T (0.1  g/L) Troponin T (0.01  g/L) OR 1.80; 1.30-2.54 1.82; 1.38-2.40 1.64; 1.31-2.06 OR 3.20; 2.22-4.592.26; 1.79-2.85 1.47; 1.12-1.93 3.42; 2.57-5.984.29; 3.02-6.09 Pos % % % % 41 15 113 139 86 25 136 197 116 36 221 301

14. CP887679- 13 Correspondence Between Commercial Assays and Western Blotting Assay Positive WBDSA Negative WBDSA Total Beckman 10% CV + 17 20 37 - 11 106 117 Roche 10% CV + 16 8 24 - 12 118 130 Beckman 99 th % + 20 38 58 - 8 88 96 Roche 99 th % + 21 44 65 - 7 82 89

15. CP887679- 14 Myeloperoxidase Predictions of Events When cTNT “Negative” CP1131968-3 NEJM 349:1600, 2003 Odds ratio Revascularization Myeloperoxidase quartile 2 Myeloperoxidase quartile 3 Myeloperoxidase quartile 4 Major adverse cardiac events Myeloperoxidase quartile 2 Myeloperoxidase quartile 3 Myeloperoxidase quartile 4 Revascularization Myeloperoxidase quartile 2 Myeloperoxidase quartile 3 Myeloperoxidase quartile 4 Major adverse cardiac events Myeloperoxidase quartile 2 Myeloperoxidase quartile 3 Myeloperoxidase quartile 4 30 Days 6 Months Unadjusted Adjusted

16. CP887679- 15 Prognostic Effects of MPO at 72 Hours CP1132618-7 Death, MI (%) Circulation 108:1443, 2003 Before PCI Adjusted hazard ratio 2.04 (0.65-6.42) Adjusted hazard ratio 3.07 (1.21-4.26) MPO high MPO low After PCI HoursHours

17. CP887679- 16 cTnI Negative ACS? Circulation 107:533, 2003 CP1104508-19

18. CP887679- 17 Sensitivity of cTnI, CK-MB, and Myoglobin with Sensitive Contemporary Assays AHJ 148:577, 2004 TnI (0.1 µg/L)CK-MB (3.5 µg/L)Myo (98/56 µg/L) CP1176222-2 0 min30 min60 min90 min Sensitivity 2 hr3 hr6 hr6 hr Specificity TnI (0.4 µg/L)TnI (0.1 µg/L)TnI (0.07 µg/L)

19. CP887679- 18 100 80 60 40 20 0 Sensitivity of Admission Values By Time from Onset of Symptoms CP1176222-4 AHJ 148:578, 2004 TnI 0.07 µg/L TnI 0.1 µg/L TnI 0.4 µg/L CK-MBMyoglobin 86 84 64 36 68 53 67 57 100 86 <4 hours (n=58) >4 hours (n=118)

20. CP887679- 19 cTnT and Angiographic Measures CP1179389-5 TnT (<0.01) Prog Cardiovasc Dis 47(3), 2004 P<0.001 Stenosis (%) TnT (>0.01) P=0.02 Thrombus (%) P=0.03 TFG 0/1 (%) P<0.001 TMPG 0/1 (%)

21. CP887679- 20 JACC 36(3):970- 1062, 2000 Medical and Interventional Response to GP IIb/IIIa Agents in Troponin Positive Patients +24h+48h  +24h+48h  +72h n=1,265 OR=0.37 P=0.032 CAPTURE 2.8% 1.3% n=9,461 OR=0.72 P=0.003 PURSUIT n=1,570 OR=0.45 P=0.016 PRISM-PLUS n=12,296 OR=0.66 P=0.001 All n=1,239 OR=0.46 P=0.009 n=1,228 OR=0.71 P=0.105 n=287 OR=0.35 P=0.062 n=2,754 OR=0.59 P=0.001 5.8% 2.8% 10.3% 7.6% 8.0% 2.9% 8.0% 4.9% 4.4% 3.2% 3.8% 1.8% 4.3% 3.9% Death or MI (%) Start GP IIb/IIIa inhibitor/placebo Percutaneous coronary intervention CP956024-13

22. CP887679- 21 LMW Heparin vs VF Heparin in TIMI 11B J Am Coll Cardiol 36:1812, 2000 CP1008642-5 Clinical events at 14 days (%) CKMB negative patients Enoxaparin UFH 6 P=0.006 6 0 4 6 9 17 10 11 40 21 28 P=0.007 cTnI Negative n=179 cTnI Positive n=180

23. CP887679- 22 TACTICS (TIMI 18) CP1036852-9 ConservativeInvasive No.treatmenttreatment Primary endpoint <0.1 ng/mL734 4.36.6 0.1 - <0.418116.54.4 0.4 - <1.521317.65.4  1.569315.68.8 Death or MI <0.1 ng/mL734 1.93.0 0.1 - <0.418112.14.4 0.4 - <1.521311.82.7  1.569310.05.9 ConservativeInvasive No.treatmenttreatment Primary endpoint <0.1 ng/mL734 4.36.6 0.1 - <0.418116.54.4 0.4 - <1.521317.65.4  1.569315.68.8 Death or MI <0.1 ng/mL734 1.93.0 0.1 - <0.418112.14.4 0.4 - <1.521311.82.7  1.569310.05.9 Cardiac troponin I JAMA 286:2405, 2001 0.010.11.010 Favors invasive treatment Favors Conservative treatment Odds ratio

24. CP887679- 23 Relation Between Creatinine Clearance, Troponin T, and Outcomes

25. CP887679- 24 CURE Rates and Relative Risks of First Primary Outcome CP1019654-1 Pt with event (%) Patients Characteristic(no.)PlaceboClopidogrel Overall12,56211.4 9.3 Associated MI 3,28313.711.3 No associated MI 9,27910.6 8.6 Male 7,72611.9 9.1 Female 4,83610.7 9.5  65 yr old 6,354 7.6 5.4 >65 yr old 6,20815.313.3 ST-segment deviation 6,27514.311.5 No ST-segment deviation 6,287 8.6 7.0 Enzymes elevated at entry 3,17613.010.7 Enzymes not elevated at entry 9,38610.9 8.8 Diabetes 2,84016.714.2 No diabetes 9,722 9.9 7.9 Low risk 4,187 6.7 5.1 Intermediate risk 4,185 9.4 6.5 High risk 4,18418.016.3 History of revascularization 2,24614.4 8.4 No history of revascularization10,31610.7 9.5 Revascularization after randomization 4,57713.911.5 No revascularization after randomization 7,98510.0 8.1 Patients Characteristic(no.)PlaceboClopidogrel Overall12,56211.4 9.3 Associated MI 3,28313.711.3 No associated MI 9,27910.6 8.6 Male 7,72611.9 9.1 Female 4,83610.7 9.5  65 yr old 6,354 7.6 5.4 >65 yr old 6,20815.313.3 ST-segment deviation 6,27514.311.5 No ST-segment deviation 6,287 8.6 7.0 Enzymes elevated at entry 3,17613.010.7 Enzymes not elevated at entry 9,38610.9 8.8 Diabetes 2,84016.714.2 No diabetes 9,722 9.9 7.9 Low risk 4,187 6.7 5.1 Intermediate risk 4,185 9.4 6.5 High risk 4,18418.016.3 History of revascularization 2,24614.4 8.4 No history of revascularization10,31610.7 9.5 Revascularization after randomization 4,57713.911.5 No revascularization after randomization 7,98510.0 8.1 Relative risk (95% CI) Clopidogrel better Placebo better NEJM 345:494, 2001

26. CP887679- 25 CP1189452-4 4 peptides related with each other regarding biochemistry and physiological function Atrial natriuretic peptide (ANP) Brain (or B type) natriuretic peptide (BNP) C-type natriuretic peptide (CNP) Urodilatin, a slightly extended form of ANP 4 peptides related with each other regarding biochemistry and physiological function Atrial natriuretic peptide (ANP) Brain (or B type) natriuretic peptide (BNP) C-type natriuretic peptide (CNP) Urodilatin, a slightly extended form of ANP H2NH2NH2NH2N —COOH—COOH C F C D RI S G L C C Natriuretic Peptide Family Natriuretic Peptide Family

27. CP887679- 26 Common 17-amino acid ring structure Ring structure highly conserved 11/17 amino acids are homologous Ring structure essential for physiological activity Common 17-amino acid ring structure Ring structure highly conserved 11/17 amino acids are homologous Ring structure essential for physiological activity H2NH2NH2NH2N —COOH—COOH C F C D RI S G L C C CP1189452-5 Natriuretic Peptide Family Biochemistry

28. CP887679- 27 CardiomyocyteCardiomyocyte BloodBlood proBNP (108 aa) BNP-32 (proBNP 77-108) proBNP (108 aa) BNP-32 (proBNP 77-108) BNP-32 Physiologically active form BNP-32 Physiologically active form NT-proBNP 1-76 Secretion LV stretch Wall tension Secretion LV stretch Wall tension Pre-proBNP (134 aa) Signal peptide (26 aa) proBNP Serin protease (Corin?) Serin protease (Corin?) Mair: Scand J Clin Lab Invest, 1999 CP1189452-10

29. CP887679- 28 BNP Values by Age and Gender JACC 40:978, 2002 Age (yr) Biosite ® BNP (pg/mL) Shionogi ® BNP (pg/mL) CP1189452-19 Males Age (yr) Females

30. CP887679- 29 CP1189452-20 97.5% percentile (pg/mL) <3030-3940-4950-59  60 Age groups Males Females Age-Specific Reference Range for NT-proBNP

31. CP887679- 30 AHJ 149(4), April 2005 pg/mLpg/mL NT-proBNPNT-proBNP pg/mLpg/mL BNPBNP Weight categories (BMI)  25 25-29.925-29.9  30  25 25-29.925-29.9  30 P<0.001P<0.001P<0.001P<0.001 Values of BNP and NTproBNP by Weight CP1189452-21

32. CP887679- 31 Influence of multiple sampling on BNP and NT-proBNP RCVs; the RCVs for BNP, NT- proBNP, and BNP >350 ng/L are shown separately, as derived from singleton, duplicate, and triplicate sampling with each sample analyzed in singleton, for estimating the homeostatic setpoints of the 2 serial results Week-to- week RCV (%) Patient samples (no.) BNP NT-proBNP BNP >350 ng/L BNP NT-proBNP BNP >350 ng/L CP1189452-22 Clin Chem 50:2054, 2004 57 82 68 113 98 69 87 64 54 Multiple Sampling 1 1 2 2 3 3

33. CP887679- 32 38 ± 4 BNP concentration (pg/mL) BNP concentration (pg/mL) Diagnosis n=139 1,076 ± 138 No CHF CP1189452-25 n=97 CHF LV dysfunction No acute CHF LV dysfunction No acute CHF n=14 141 ± 31 BNP Levels of Patients Diagnosed Without CHF, with Baseline Left Ventricular Dysfunction, and with CHF

34. CP887679- 33 BNP Levels in Heart Failure Median BNP level (pg/mL) Median BNP level (pg/mL) CP1189452-26 BNP Levels in Normal Subjects and Inpatients with Heart Failure Normal I I II III IV Class

35. CP887679- 34 CP1189452-27 ED Probability of CHF Recorded P<0.0001 from clinical judgment to combined McCullough PA et al: Circulation 106:416, 2002 Diagnostic accuracy (%) Clinical judgment BNP Combined 70727476788082 74.0 81.2 81.5 n=1,538 BNP Study Primary End Point

36. CP887679- 35 n=1,586;  50% CHF; 56% male; 64±17 yo; 41% COPD;  30% Hx CHF CP1189452-28 BNP (pg/mL)SensSpecPPVNPV 5097627195 10090767989 15085838385 BNP (pg/mL)SensSpecPPVNPV 5097627195 10090767989 15085838385 AUC = 0.91 Breathing Not Proper (BNP) Multicenter Trial

37. CP887679- 36 Sensitivity (true-positives) 1-specificity (false-positives) AJC 95, April 15, 2005 Area under the curve Combined0.96 NT-proBNP0.94 Clinical judgment0.90 Area under the curve Combined0.96 NT-proBNP0.94 Clinical judgment0.90 ModalityModality NT-proBNP and Clinical Judgment of the Diagnosis of CHF CP1189452-29

38. CP887679- 37 AJC 95, April 15, 2005 Optimal cut-point SensSpecPPVNPVAccuracy (%) (%)(%)(%)(%) (%) Rule-in cutpoints All pt (n=599)9009085769487 <50 yr old4509395679995 n=144  50 yr old9009180779285 n=455 Rule-out pt All pt (n=599) 3009968629983 Optimal cut-point SensSpecPPVNPVAccuracy (%) (%)(%)(%)(%) (%) Rule-in cutpoints All pt (n=599)9009085769487 <50 yr old4509395679995 n=144  50 yr old9009180779285 n=455 Rule-out pt All pt (n=599) 3009968629983 CP1189452-30 Optimal NT-proBNP Cutpoints for Acute Congestive Heart Failure

39. CP887679- 38 McCullough PA et al for the BNP Multinational Study Investigators: J Am Coll Cardiol 41:278A, 2003 Application of BNP Testing in CHF “Grey Zone” BNP BNP (pg/mL) “Grey Zone” BNP 100-500 pg/mL 26.4% of all cases 16.5% CHF 7.9% No CHF 0 0 100 200 300 400 500 600 700 800 900 1,000 1,100 1,200 1,300 CP1189452-31

40. CP887679- 39 CP1189452-32 pg/mL Coronary pulmonale200-500 Primary pulmonary300-500 hypertension Acute pulmonary150-500 embolism pg/mL Coronary pulmonale200-500 Primary pulmonary300-500 hypertension Acute pulmonary150-500 embolism BNP Elevations Right-Sided Coronary Heart Failure

41. CP887679- 40 CP1189452-33 Acute or chronic systolic or diastolic HF LV hypertrophy Inflammatory cardiac diseases Systemic arterial hypertension with LVH Pulmonary hypertension Acute or chronic renal failure Ascitic liver cirrhosis Endocrine disorders (eg, hyper- aldosteronism, Cushing’s syndrome) Acute or chronic systolic or diastolic HF LV hypertrophy Inflammatory cardiac diseases Systemic arterial hypertension with LVH Pulmonary hypertension Acute or chronic renal failure Ascitic liver cirrhosis Endocrine disorders (eg, hyper- aldosteronism, Cushing’s syndrome) When Can Non-CHF Patients Present with BNP Elevations?

42. CP887679- 41 CP1189452-34 Well HF patients Acute mitral regurgitation Pulmonary edema <1 hour old Other cases “up-stream” from left ventricle Mitral stenosis Atrial myoxma Well HF patients Acute mitral regurgitation Pulmonary edema <1 hour old Other cases “up-stream” from left ventricle Mitral stenosis Atrial myoxma When Will CHF Present Without BNP Elevations?

43. CP887679- 42 Circulation 108:278, 2003 Cumulative probability of death (%) DaysDays >1,869 ng/L P<0.001P<0.001 669-1,869 ng/L 238-668 ng/L  237 ng/L CP1189452-43 Survival – GUSTO IV ACS

44. CP887679- 43 1 1 2 2 3 3 4 4  in risk Inclusion Day 2 6 weeks 3 months 6 months  in risk Inclusion Day 2 6 weeks 3 months 6 months JACC 45(4), 2005 1.36 (1.04-1.76) 1.83 (1.23-2.72) 1.89 (1.14-3.14) 1.29 (0.99-1.67) 1.46 (1.06-2.03) CP1189452-44 Odds Ratio for Events by Time <URL  URL<HC  HC 1.85.02.57.9 2.05.33.37.2 0.63.81.96.4 0.53.41.28.5 0.22.10.75.5 <URL  URL<HC  HC 1.85.02.57.9 2.05.33.37.2 0.63.81.96.4 0.53.41.28.5 0.22.10.75.5  in risk

45. CP887679- 44 P < 0.0001P = 0.9 Baseline BNP & Clinical Outcomes P < 0.0001P = 0.9 DeathMI N = 1356 N = 320 Morrow DA JACC 2004

46. CP887679- 45 CP1173030-5 Circulation 110:3206, 2004 Death and AMI by NTproBNP Levels 30-day follow-up Event rate at 30 days (%) 30-day follow-up Death Myocardial infarction Odds ratio 6.0 [2.7-13.5] Odds ratio 6.0 [2.7-13.5] NT-proBNP low NT-proBNP high Odds ratio 2.6 [1.5-4.5] Odds ratio 2.6 [1.5-4.5] NT-proBNP low NT-proBNP high

47. CP887679- 46 CP1173030-6 Relationship of NT-proBNP and cTnT *P<0.01 vs NT-proBNP ≤250 ng/L Circulation 110:3206, 2004 *P<0.01 vs NT-proBNP ≤250 ng/L Circulation 110:3206, 2004 15.1 7.2 9.1 7.1 5.6 1.3 >250 ng/L≤250 ng/L <0.01 μg/L 0.01 - 0.1 μg/L >0.1 μg/L NT-proBNP Troponin T Death, myocardial infarction (%) * * * *

48. CP887679- 47 Death/MI at 6 months Test for interaction: P=NS % % BNP neg BNP cut point = 80 pg/mL CP1189452-45 CONS INV BNP pos (19% BNP pos) n=681685156164 Morrow DA: JACC, 2004 Baseline BNP: CONS vs INV Strategy

49. CP887679- 48 Beta-Blocker Therapy and BNP Levels Luchner A et al: JACC 32:1839, 1998 pg/ mL pmol/ mL ANP  32% ANP  32% BNP  89% BNP  89% cGMP  18% cGMP  18% n=592 n=80 P<0.01 No  -blocker (n=592)  -blocker (n=80) No  -blocker (n=592)  -blocker (n=80) CP1189452-59

50. CP887679- 49 Outcomes by Number of Positive Outcomes TACTICS-TIMI 18 CP1143360-10 Circ 109:584, 2004 Death or MI (%) Female Male Female Male P=NS Positive markers (no.) P=NS 2.9 4.4 6.2 6.5 12.1 13.3 Death or MI by Multimarker Approach

51. CP887679- 50 Odds of Positive Marker TACTICS-TIMI 18 CP1143360-7 Circ 109:583, 2004 T18 Tnl T11 Tnl Combined T18 TnT T11 TnT Combined T18 BNP T16 BNP Combined T18 CRP T11 CRP Combined T18 Tnl T11 Tnl Combined T18 TnT T11 TnT Combined T18 BNP T16 BNP Combined T18 CRP T11 CRP Combined Male more likely Female more likely 00.511.52

52. CP887679- 51 Outcomes by Gender TACTICS-TIMI 18 CP1143360-11 Circ 109:584, 2004 Any Marker Positive (n=442) Women No Marker Positive (n=173) Men % % Invasive Conser- vative 30 20 10 0 P=0.03 P=0.02 Rehosp Death/MI Any Marker Positive (n=885) No Marker Positive (n=297) Invasive Conser- vative P=0.002 P=NS P=0.07 P=NS